Twenty-one cytotoxic T lymphocyte (CTL) clones or lines that killed autologous tumor cells, but not allogeneic tumor, K562, or Daudi cells, were established from fresh tumor-infiltrating lymphocytes of two individuals (HP-1 and HP-2) with head and neck cancer by limiting dilution in the presence of recombinant interleukin-2. Sixteen (76%) of these 21 clones or lines comprised CD4+ CTLs and the other five comprised CD8+ CTLs. These observations suggest that autologous tumor cell-specific CD4+ CD8- and CD4- CD8+ CTLs are present in vivo at the tumor site in head and neck cancer. Analysis of T cell receptor (TCR) gene arrangements in 20 of the 21 CTL isolates with reverse transcriptase and the polymerase chain reaction revealed that five of 12 and five of eight isolates from HP-1 and HP-2, respectively, were clones, the other isolates being lines comprised of two or more clones. Each CTL clone showed a different combination of V alpha and V beta gene expression, suggesting that more than five different tumor-associated antigens may be expressed on head and neck cancer cells. In spite of the diversity of TCR alpha beta combinations, TCR V alpha 1, V alpha 3, V alpha 8, V alpha 10, V beta 8, V beta 9, and V beta 17 were also frequently expressed in both patients. These data suggest that specific CTLs proliferate oligoclonally and contribute to the specific immune response against head and neck cancer in vivo.